Description of the Prior Art
A description of media and procedures for identifying certain members of Class Mollicutes is provided by G. Masover and L. Hayflick, "The Genera Mycoplasma, Ureaplasma, and Acholeplasma, and Associated Organisms (Thermoplasmas and Anaeroplasmas), The Prokaryotes, Vol. II, Springer-Verlag, New York (1981), the entire contents of which are hereby incorporated by reference.
Sampling, inoculation and incubation procedures and growth medium formulations are also described by G. E. Kenney in the Manual of Clinical Microbiology, 3rd edition, American Society of Microbiology, Washington, D.C. (1980), 365-370. A variety of prior art techniques are described. Cultivation in a nutrient broth, and pipette transfer of broth to an agar medium is described. Use of a diphasic medium, that is, an agar layer in the bottom of a test tube covered by a liquid medium is also described. The procedures described in the Manual are representative of the procedures often followed prior to this invention. Three agar plates, a diphasic system and a broth are inoculated with specimen or sample. Each agar plate is incubated under different conditions, one aerobically, another microaerophilically and the third under strict anaerobiosis. The diphasic culture is also incubated under strict anaerobiosis (Kenny, p. 369, supra). Subcultures from the broth to the agar plates and diphasic system are made on the fourth day after incubation of the broth with sample and incubated for an additional 3 to 5 days prior to assessing results. Thus a total of nine cultures are made for a single sample. Despite these complex procedures, the results are often unreliable, even when carried out by a skilled microbiologist due to interlaboratory variations in the microenvironment in which the tests are carried out.
Triphasic systems for blood cultures are described in U.S. Pat. Nos. 2,992,974 and 3,589,983. In the procedures of both patents, simultaneous inoculation of liquid and solid nutrient media with a blood sample is described, and the incubation occurs with the media being separated. U.S. Pat. No. 4,308,347 is directed to a dual container combination wherein a specimen is incubated in a liquid medium in one container and the container is opened, exposing it to the atmosphere, and connected to a second container containing a solid medium. The liquid contents are transferred to the solid medium and incubated in a diphasic mode. The devices disclosed in the above patents are not suitable for carrying out the method of this invention, in particular microscopic examination of the solid media in the unopened device, and the methods described therein are are very different from those of this invention.
U.S. Pat. No. 3,449,210 describes a microorganism culturing assembly which includes a transparent bottle and cap which can be used with solidified culture media. The shape and relative dimensions of the bottle are not suitable for microscopic examination of the contents in accordance with this invention. U.S. Pat. No. 3,651,926 describes a transport system for biological specimens, and U.S. Pat. No. 3,904,482 describes a blood sample cultivation system.